treatment of cerebellar organotypic cultures with dna damaging agents for fluorescent imaging of par chains to assess genotoxic stress response

<meta charset="utf8"></head><body>小脑浦肯野细胞：器官型培养物中的 DNA 损伤反应

The integrity of cellular DNA is constantly under threat from DNA damaging agents, predominantly metabolic by-products like reactive oxygen species, which inflict tens of thousands of DNA lesions per cell daily1. The persistent upkeep of genome stability is essential for cellular homeostasis2,3. The cornerstone of this maintenance is the DNA damage response (DDR) - an intricate, layered signaling network that initiates specific DNA repair pathways while carefully adjusting many other cellular processes4,5. Deficits in the DDR are commonly manifested as &#39;genome instability syndromes,&#39; marked by chromosomal instability, progressive tissue deterioration, impaired growth or development, a predisposition to cancer, and heightened sensitivity to particular DNA-damaging agents6,7,8,9. Notably, neurodegeneration, which often includes cerebellar atrophy, is a distinct feature of many genome instability syndromes7,10,11,12.
The autosomal recessive disorder, ataxia-telangiectasia (A-T), is a well-documented example of a genome instability disorder13,14,15. This condition arises from null mutations in the ATM (A-T, mutated) gene, responsible for coding the pivotal protein kinase, ATM, which is known primarily as a DDR mobilizer in response to DNA double-strand breaks (DSBs)16,17. A-T manifests as a multisystem disorder, predominantly characterized by progressive cerebellar degeneration, leading to acute motor impairments, immunodeficiency, gonadal atrophy, cancer predisposition, and extreme sensitivity to ionizing radiation. Cultured cells from individuals with A-T show chromosomal instability and increased sensitivity to genotoxic agents, especially those causing DSBs15,18,19. Importantly, ATM also plays a role in repairing other DNA lesions, underscoring its broad significance in maintaining genome stability20,21,22.
Despite thorough research into ATM&#39;s numerous roles, the specific mechanism leading to cerebellar degeneration in A-T remains a topic of active debate, with various models proposed to elucidate this process23,24,25,26,27,28,29,30. Our model28 suggests that cerebellar degeneration in A-T patients begins with the dysfunction and eventual loss of Purkinje cells (PCs). Considering ATM&#39;s critical role in preserving genome stability in the face of ongoing DNA damage, PCs are particularly vulnerable to the absence of ATM. We attribute this vulnerability to the combination of their high metabolic activity, distinctive chromatin structure, and extensive transcriptional activity. Ultimately, it is suggested that the loss of PC function, and hence, their degeneration, is due to the stochastic, functional inactivation of genes, a consequence of producing defective transcripts28.
The study of PC biology in the laboratory is impeded by challenges in cultivating isolated PCs, as these cells rely heavily on their natural milieu and neighboring cells for survival and function, rendering them incompatible with dissociated culture growth. Nonetheless, PCs can remain viable for extended periods in tissue slice cultures. Cerebellar organotypic cultures, which are tissue slices typically derived from rodent cerebella, maintain the tissue&#39;s structural organization and support various experimental manipulations analogous to those possible with cultured cells. Therefore, these cultures allow for cerebellar studies within a controlled setting31,32,33,34,35,36,37,38,39,40,41,42,43. Specifically, in the context of A-T, murine cerebellar organotypic cultures have proven to be instrumental in exploring the DDR in Atm-deficient PCs40,41,42,43. While Atm-deficient mice display only a subtle cerebellar phenotype44,45,46,47, presumably due to differences between human and mouse cerebellar physiology, the assumption is that ATM&#39;s roles are largely conserved across these species. This notion is supported by our observations that the deficient response to DNA DSBs in Atm-/- murine PCs aligns with that observed in other murine and human ATM/Atm-deficient cell types40,41,42,43.
A limitation in analyzing PC responses to various stimuli or stresses within organotypic cultures is the necessity to rely on microscopic imaging for readouts. The DDR is usually studied using bulk biochemical readouts, although common immunofluorescent markers are utilized as well, such as following the dynamics of formation and resolution of nuclear foci of phosphorylated histone H2AX (&#947;H2AX) and the 53BP1 protein, which are considered indicators of DSBs48,49. A broader measure is the fluorescent imaging of poly(ADP-ribose) (PAR) chain formation on proteins, a rapid and robust early DNA damage response, particularly to strand breaks7,50. We modified the protocol by Komulainen et al.51 for PAR staining in cerebellar organotypic cultures. We observed a pronounced PAR response in the sizeable nuclei of PCs. Presented here is our refined protocol for establishing murine cerebellar organotypic cultures and for visualizing the PAR response under genotoxic stress.

<meta charset="utf8"></head><body>作者聚焦：破译 ATM 在共济失调-毛细血管扩张症和相关小脑变性中的作用

使用小脑器官型培养物可视化浦肯野细胞中的 DNA 损伤反应

用 DNA 损伤剂处理小脑器官型培养物，用于 PAR 链的荧光成像以评估遗传毒性应激反应

treatment-cerebellar-organotypic-cultures-with-dna-damaging-agents

Research

JoVE Journal

Biology

Treatment of Cerebellar Organotypic Cultures with DNA Damaging Agents for Fluorescent Imaging of PAR Chains to Assess Genotoxic Stress Response

151 次观看. 首先，从基础培养基鹰中的 10 日龄小鼠幼崽中回收的小脑组织进行培养。将 DNA 损伤化学物质以适当的最终浓度直接添加到培养基中。暴露期后，用新鲜制备的含有 DNA 损伤化学物质和聚 ADP 核糖糖水解酶抑制剂的混合培养基更换培养基，并孵育 30 分钟。30 分钟后，在室温下用 0.1 摩尔磷酸盐缓冲液洗涤切片，并立即用预冷的丙酮和甲醇混合物?...

视频: 用 DNA 损伤剂处理小脑器官型培养物，用于 PAR 链的荧光成像以评估遗传毒性应激反应

Visualizing the DNA Damage Response in Purkinje Cells Using Cerebellar Organotypic Cultures

Cerebellar Purkinje cells (PCs) exhibit a unique interplay of high metabolic rates, specific chromatin architecture, and extensive transcriptional activity, making them particularly vulnerable to DNA damage. This necessitates an efficient DNA damage response (DDR) to prevent cerebellar degeneration, often initiated by PC dysfunction or loss. A notable example is the genome instability syndrome, ataxia-telangiectasia (A-T), marked by progressive PC depletion and cerebellar deterioration. Investigating DDR mechanisms in PCs is vital for elucidating the pathways leading to their degeneration in such disorders. However, the complexity of isolating and cultivating PCs in vitro has long hindered research efforts. Murine cerebellar organotypic (slice) cultures offer a feasible alternative, closely mimicking the in vivo tissue environment. Yet, this model is constrained to DDR indicators amenable to microscopic imaging. We have refined the organotypic culture protocol, demonstrating that fluorescent imaging of protein-bound poly(ADP-ribose) (PAR) chains, a rapid and early DDR indicator, effectively reveals DDR dynamics in PCs within these cultures, in response to genotoxic stress.

Cerebellar Purkinje cells (PCs) are particularly sensitive to deficiencies in the DNA damage response. A protocol is presented for the visual evaluation of the dynamics of PC response to DNA damage, which involves staining protein-bound poly(ADP-ribose) chains within cerebellar organotypic cultures.